Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry.

A sensitive method for the identification and quantification of anabolic steroids and clenbuterol at trace levels in dietary supplements by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in atmospheric pressure ionisation (APCI) mode using a single-stage Orbitrap analyser operating at a resolution power of 100 000 full width at half maximum (FWHM) was developed and validated. A total of 1 g of dietary supplement was added with testosterone-d3 as internal standard, dissolved in methanol, evaporated to dryness, diluted in sodium hydroxide solution and extracted with a mixture of pentane/ethyl ether 9:1. The extract was directly injected into the LC-HRMS system. The method was fully validated. Limits of detection (LODs) obtained for anabolic androgenic steroids (AASs) varied from 1 to 25 ng g(-1) and the limit of quantitation (LOQ) was 50 ng g(-1) for all analytes. The calibration was linear for all compounds in the range from the LOQ to 2000 ng g(-1), with correlation coefficients always higher than 0.99. Accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Good values of matrix effect and recovery were achieved. The ease of the sample preparation together with a fast run time of only 16 min permitted rapid identification of the analytes. The method was applied to the analysis of 30 dietary supplements in order to check for the presence of anabolic agents not labelled as being present in these supplements. Many AASs were often detected in the same sample: indeed, androstenedione was detected in nine supplements, 5-androsten-3ß-ol-17-one (DHEA) in 12, methandienone in three, stanozolol in one, testosterone in seven and testosterone esters in four of them. A retrospective analysis of suspected compounds not included at the beginning of the method development was also possible by means of the full acquisition spectra obtained with the HRMS technique.

Effects of three different medications on metabolic parameters and testicular volume in patients with hypogonadotropic hypogonadism: 3-year experience.

INTRODUCTION: The aim of this study was to demonstrate the influences of three different treatment strategies on biochemical parameters and testicular volume (TV) in patients with idiopathic hypogonadotropic hypogonadism (IHH). SUBJECTS DESIGN AND METHODS: Seventy-seven never-treated patients with IHH and age and body mass index (BMI)-matched 42 healthy controls were analysed in a retrospective design. Twenty-eight patients were treated with testosterone esters (TE), 25 patients were treated with human chorionic gonadotropin (hCG) and 24 patients were treated with testosterone gel (TG). Biochemical parameters, tanner stages (TS) and TV were evaluated before and after 6 months of treatment. RESULTS: Pretreatment TV, TS and biochemical test results were similar among the three treatment subgroup. In the TE-treated group, BMI, haemoglobin, haematocrit, creatinine, triglyceride, total testosterone (TT), TS and TV increased, but HDL-cholesterol (C) and urea level decreased significantly. In the hCG-treated group, triglyceride level decreased, and luteinizing hormone level, TS and TV increased significantly. BMI, TT, TS and TV increased, and leucocyte count, total-C, HDL-C levels decreased significantly in the TG-treated patients. No treatment type resulted in any changes in insulin resistance markers. CONCLUSION: hCG treatment resulted in favourable effects particularly on TV and lipid parameters. When TV improvement is considered less important, TG treatment may be a better option for older patients with IHH because of its easy use, neutral effects on triglyceride, haemoglobin and haematocrit, and its beneficial effects on total cholesterol level.

Hepatocellular adenomas associated with anabolic androgenic steroid abuse in bodybuilders: a report of two cases and a review of the literature.

Anabolic androgenic steroids (AAS) are used illicitly at high doses by bodybuilders. The misuse of these drugs is associated with serious adverse effects to the liver, including cellular adenomas and adenocarcinomas. We report two very different cases of adult male bodybuilders who developed hepatocellular adenomas following AAS abuse. The first patient was asymptomatic but had two large liver lesions which were detected by ultrasound studies after routine medical examination. The second patient was admitted to our hospital with acute renal failure and ultrasound (US) studies showed mild hepatomegaly with several very close hyperecogenic nodules in liver, concordant with adenomas at first diagnosis. In both cases the patients have evolved favourably and the tumours have shown a tendency to regress after the withdrawal of AAS. The cases presented here are rare but may well be suggestive of the natural course of AAS induced hepatocellular adenomas. In conclusion, sportsmen taking AAS should be considered as a group at risk of developing hepatic sex hormone related tumours. Consequently, they should be carefully and periodically monitored with US studies. In any case, despite the size of the tumours detected in these two cases, the possibility of spontaneous tumour regression must also be taken in account.

Treatment of constitutional delayed puberty with a combination of testosterone esters.

Thirteen boys who had constitutional delayed puberty (CDP) were treated with a combination of short- and long-acting testosterone esters (testosterone propionate, testosterone phenylpropionate, testosterone isocaproate). Mean age at the onset of treatment was 14.9 +/- 0.6 years and bone age delay was -2.7 +/- 0.9 years. An intramuscular dose of 200 mg testosterone was administered 4 times at 3-week intervals and the treated CDP boys were followed for 2 years. All boys with CDP entered puberty after the last dose (testicular volume > or = 4 ml) and growth rate increased from 4.5 +/- 0.5 cm/year pretreatment to 8.4 +/- 1.6 cm/year posttreatment after the 2-year follow-up period. Height for bone age SD score did not alter significantly from a mean of -1.1 pretreatment to -1.3 posttreatment as well as predicted height pretreatment (173.5 +/- 6.6 cm) and posttreatment (173.3 +/- 4.9 cm). A combination of testosterone esters in a given dose and schedule is a safe and effective treatment for prepubertal boys with CDP.

Effects of testosterone on the rat renal medullary vasopressin receptor concentration and the antidiuretic response.

The renal concentrating ability declines with age in humans and animals. Studies suggest that the concentrating defect is due to a decrease in renal vasopressin sensitivity. With ageing, expression of the renal vasopressin V2 receptor in rat is impaired; the normal receptor expression is restored by testosterone treatment. The effect of testosterone on the renal sensitivity to vasopressin was investigated in young rats. Male rats after orchidectomy and chronic antiandrogen cyproterone acetate treatment, and female rats after chronic testosterone phenylpropionate treatment, were used. The plasma arginine-vasopressin (AVP) and testosterone concentrations, and the antidiuretic responses to AVP and the V2 agonist deamino-[8-D-arginine]-vasopressin (dDAVP) after volume loading were measured, and the renal [3H]AVP binding density was determined. The plasma AVP level decreased slightly, but not significantly, in male rats after orchidectomy and cyproterone acetate treatment, but did not alter in female rats after testosterone treatment. The AVP and dDAVP sensitivities decreased in male rats after orchidectomy and cyproterone acetate administration, and increased in female rats treated with testosterone, as compared with the animals with a normal gonadal function. [3H]AVP binding to the renal inner medullary membranes was decreased following orchidectomy or antiandrogen treatment in male rats, and increased in testosterone-treated female rats. The results suggest that testosterone may play a physiological role in maintenance of the V2 vasopressin receptor expression and hence in the normal urinary concentrating ability in rat.

Treatment of constitutional delayed puberty with a combination of testosterone esters.

Thirteen boys with constitutional delayed puberty (CDP) were treated with a combination of short and long-acting testosterone esters (testosterone propionate, testosterone phenylpropionate, testosterone isocaproate). Mean age at the onset of treatment was 14.9 +/- 0.6 years and bone age delay was -2.7 +/- 0.9 years. The dose of testosterone used was 200 mg intramuscularly four times at three week intervals, and the treated CDP boys were followed for two years. All the boys with CDP entered puberty after the last dose (testicular volume > or = 4 ml), and growth rate increased from 4.5 +/- 0.5 cm/year, pretreatment, to 8.4 +/- 1.6 cm/year, posttreatment, at the two year follow-up. Height for bone age SD score did not change significantly from a mean of -1.1 before treatment to -1.3 after treatment, nor did predicted height before treatment (173.5 +/- 6.6 cm) and after treatment (173.3 +/- 4.9 cm). Combination of testosterone esters in a given dose and schedule is a safe and effective treatment for prepubertal boys with constitutional delayed puberty.

Structural determination of testosterone esters in oil injectables by thermospray mass spectrometry.

In the absence of analytical reference standards, the combination of thermospray and collision-activated dissociation mass spectrometry has been utilized to separate and identify the presence of several esters of testosterone in imported oil injectables. Structural identifications were based on generic daughter ions considered diagnostic of the steroidal ring structure of these anabolic steroids as well as daughter ions representative of the ester alkyl or aryl side chains.

[Cytogenetic effect of testosterone on rat hepatocytes]. / Tsitogeneticheskoe vliianie testosterona na kletki pecheni krys.

The capacity of testosterone administered in vivo in the form of testosterone propionate (TP), to modify chromosome integrity and a search for certain conditions under which the hormone manifested cytogenetic activity, were determined. TP was administered at a single dose or for 12 days at various doses to random bred albino male rats aged 3-4.5 mos. Single administration of the hormone to animals aged 4-4.5 mos. did not result in an increase in the number of hepatocytes with chromosome aberrations even at a dose exceeding 6-7-fold daily production. However after daily administration for 12 days at a dose exceeding only twice the daily one, TP increased 1.5-fold the number of cells with damaged chromosomes. In animals aged 3-3.5 mos. the same scheme of administration of TP turned out ineffective. The results obtained are under discussion.

[Morphological characteristics of the uterine reaction of androgenized rats in the neonatal period to the administration of sex steroids]. / Morfologicheskie kharakteristiki reaktsii matki neonatal'no androgenizirovannykh krys na vvedenie polovykh steroidov.

In female rats subjected to early neonatal androgenization (50 micrograms of testosterone propionate) the uterus at the age of maturity is of normal sizes and mass whereas its hydro-structure is substantially changed: the content of stromal elements is increased and the number of glands in the endometrium is decreased. The endometrium of androgenized female rats is not sensitive to progesterone influence manifested in the absence of the collagenolytic effect of progesterone and the stimulation of uterine gland formation. It may determine stable sterility of neonatally androgenized female rats after restoration of ovulation in them.

Development of a gas chromatographic-mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroids in horse urine. I. Detection of testosterone phenylpropionate administrations to equine male castrates.

A gas chromatographic-mass spectrometric (GC-MS) method using three analytes to detect and confirm the administration to equine male castrates of veterinary pro-drugs based upon esters of testosterone is described. The method involves extraction of steroid conjugates from horse urine by C18-bonded cartridges and fractionation into glucuronic acid and sulpho-conjugates by Sephadex LH-20 column chromatography. After deconjugation, the free neutral steroids were partially purified by thin-layer chromatography and following derivatization (methyloxime-trimethylsilyl ether) were analysed by capillary GC-MS in the selected-ion or full-scan mode. Of the three analytes, 5 alpha-androstane-3 beta, 17 alpha-diol could be detected in the glucuronic acid fraction for about ten days and 5 alpha-androstane-3 beta,17 beta-diol and testosterone could be detected in the sulpho-conjugate fraction for up to nineteen days after administration of a single therapeutic dose (50 mg) of testosterone phenylpropionate to cross-bred and thoroughbred castrated male horses. The reasons for development of such a method, its validation and its potential for the detection of neutral metabolites of other veterinary anabolic steroids in horse urine are discussed.

The correlation between the degree of brain masculinization and song quality in estradiol treated female zebra finches.

Female zebra finches can be induced to develop a male-like song system by being exposed to a steroid as nestlings. After subsequent testosterone treatment they develop song whose quality depends on age and duration of early steroid treatment. This study compares the anatomy of two song nuclei with the birds' song quality. The capacity for song correlates with the volumes of the nucleus hyperstriatum, ventralis, pars caudalis (HVc) and the nucleus robustus archistriatalis (RA) and with the size of RA neurons.

Isolation and characterisation of genomic and cDNA clones for an androgen-regulated secretory protein of rat seminal vesicles.

Testosterone controls the synthesis of seminal vesicle protein F in male rats by regulating the cellular concentration of its mRNA (mRNAF). Phage lambda recombinants have been isolated containing the complete F gene. In addition plasmids have been constructed containing cDNAF sequences some of which are probably full-length (approximately 700 bp). Detailed restriction mapping shows that the F gene is 1.7 kbp long and contains approximately 1.0 kbp of intervening sequence arranged in at least two introns (420 bp and 600 bp). Part of cDNAF has been sequenced showing that the terminal 125 bp of the 3' untranslated region of mRNAF has substantial (greater than 70%) sequence homology with the 3' end of the mRNA coding for another androgen-dependent seminal vesicle protein (protein S). The cloned F gene has been detected in liver and seminal vesicle DNA along with an homologous but structurally different gene. The hormonal control of mRNAF was examined with cDNAF. A pronounced (approximately 3000-fold) differential response to testosterone was observed.